The purpose of this investigation was to characterize the nature of peptide histidine isoleucine (PHI) and vasoactive intestinal polypeptide (VIP) receptors, and to examine the role of PHI in internal anal sphincter (IAS) relaxation. The studies were performed on opossums anesthetized with alpha-chloralose. The pressures in the IAS were recorded using continuously perfused catheters. The IAS responses to PHI analogues, PHI-27, PHM-27, PHI-(14-27)-NH2, PHI-(1-13), to VIP, to rectal balloon distention, sacral nerve stimulation, and local intramural stimulation were evaluated before and after PHI-(14-27)-NH2, PHI tachyphylaxis, and the VIP antagonists [4 Cl-D-Phe6, Leu17] VIP (VIP analogue) and (N-Ac-Tyr1, D-Phe2)-GRF(1-29)-NH2 (growth hormone releasing factor analogue). The inhibitory responses by all of the PHI analogues and VIP were not modified by tetrodotoxin. PHI-(14-27)-NH2 and PHI tachyphylaxis caused significant antagonism of the fall in internal anal sphincter pressure by PHI-27 and PHM-27 without modifying the IAS responses to VIP and rectal balloon distention, sacral nerve stimulation, and local intramural stimulation. On the other hand, VIP and growth hormone releasing factor analogues caused significant antagonism of VIP responses without modifying the responses to PHI-27. We conclude that distinct PHI and VIP receptors are present in the IAS smooth muscle and that PHI may not play a significant role in the IAS relaxation via the rectoanal reflex.